Anticoagulant

ABSTRACT

The present invention provides anticoagulant piscine gill glucosaminoglycans, high affinity anticoagulant piscine glycosaminoglycans anticoagulant glycosaminoglycans extractable from salmon guts, skin, tail or gills and salts and derivatives thereof.

[0001] The present invention relates to anticoagulant piscineglucosaminoglycans, compositions containing anticoagulant piscineglucosaminoglycans, equipment coated with anticoagulant piscineglucosaminoglycans, preparation of anticoagulant piscineglucosaminoglycans, uses of anticoagulant piscine glucosaminoglycans andmethods of treatment with anticoagulant piscine glucosaminoglycans.

[0002] Glycosaminoglycans consist of two sub-groups namely,galactosaminoglycans and glucosaminoglycans.

[0003] Heparin is the name given to a class of sulphatedglucosaminoglycans having anticoagulant properties. Heparin is widelyused medically both as a coating agent for invasive medical equipment,e.g. catheters and implants, and as a therapeutic or prophylactic agent.Besides heparin, other anticoagulant sulphated glucosaminoglycans (oftenreferred to as heparinoids) are known, e.g. heparan sulphate. These toohave been used to achieve anti-coagulant or anti-opsonization effects.However heparin is the most commercially significant of the group.

[0004] The anticoagulant glucosaminoglycans are polysaccharides withrepeating sulphated disaccharide units. The polysaccharide structure mayadditionally contain other oligosaccharide substructures, e.g. thepentasaccharide unit known to bind to antithrombin (see Casu et al.,Seminars in Thrombosis and Hemostasis 25 (Suppl. 3): 17-25 (1999) andLindahl et al., J. Biol. Chem. 258: 9826-9830 (1983)). Thus besides itstrisulphated disaccharide repeat unit, heparin contains additionalsaccharide units, e.g. disulphated disaccharides, and some heparincontains the pentasaccharide which is a high affinity binding site forantithrombin. Heparin containing this pentasaccharide binding site forantithrombin is known as high affinity heparin.

[0005] The different glucosaminoglycans differ in the inter-saccharidebonds and the saccharide ring substitution. Moreover for a particularanimal species the chain length varies and thus the glucosaminoglycanshave molecular weight distributions rather than specific molecularweights, i.e. they are polydisperse.

[0006] Currently, mammalian tissue, especially from pigs, oxen andsheep, is the source for heparin. The isolation and purification ofheparin from mammalian tissue is described for example in U.S. Pat. No.2,884,358, U.S. Pat. No. 2,989,438, U.S. Pat. No. 3,016,331, Roden etal., Methods Enzymol. 26: 73 (1972), U.S. Pat. No. 4,119,774, U.S. Pat.No. 4,122,250 and WO99/03893 and in the 1979 patent publications ofPharmacia and Kabi.

[0007] Besides natural heparins, there has recently been much interestin the so-called low molecular weight heparins (LMWHs). These are saltsof sulphated glucosaminoglycans having an average molecular weight below8 kD with at least 60% mol. having a molecular weight below 8 kD. Theseare prepared by fractionation or depolymerization of natural mammalianheparin which typically may have molecular weights in the range 5 to 40kD. Commercially available LMWHs include for example salts of ardeparin,certoparin, enoxaparin, nadroparin, parnaparin, reviparin, dalteparinand tinzaparin. (See Linhardt et al., Seminars in Thrombosis andHemostasis 25 (Suppl. 3): 5-16 (1999)).

[0008] Mammalian derived natural products however have several drawbacksfor use with humans, especially where, as with heparins and heparinoids,the use involves invasive administration, e.g injection, insertion intotissue or blood vessels, or application during surgery. In particularthere is concern that mammalian derived natural products may becontaminated with infectious agents such as bacteria, viruses and prionproteins.

[0009] We have now surprisingly found that glucosaminoglycans havingexcellent anticoagulant properties can be extracted from fish gills andthat accordingly anticoagulant glucosaminoglycans for medical use may beisolated from fish gills.

[0010] Viewed from one aspect therefore the present invention providesanticoagulant piscine gill glucosaminoglycan and salts and derivatives,especially physiologically tolerable derivatives, thereof, preferably incell-free form, also preferably in sterile form, more preferably in atleast substantially pure form (e.g. containing no more than 10% byweight, preferably no more than 2% wt, of non-glucosaminoglycanbiological materials). By biological materials is meantcarbon-containing materials naturally present in the fish and having amolecular weight in excess of 500 D. Typically the glucosaminoglycanwill have a molecular weight in the range of 500 to 30,000 Daltons,preferably at least 1000 Daltons, particularly preferably at least 1500Daltons.

[0011] Viewed from a further aspect the invention provides a method ofproducing anticoagulant glycosaminoglycan comprising extractingendogenous anticoagulant glycosaminoglycan from animal material andoptionally depolymerizing and/or molecular weight fractionating saidendogenous anticoagulant glycosaminoglycan, characterised in that theanimal material is fish gill material.

[0012] The production of anticoagulant high affinity glycosaminoglycans,in particular glucosaminoglycans, from fish (i.e. fin fish), especiallysalmon, is also novel and inventive and forms a further aspect of theinvention. Not all glucosaminoglycans or even glycosaminoglycans havethe pentasaccharide binding site for antithrombin; indeed generally lessthan half do have such sites. Thus viewed from this aspect the inventionprovides high affinity anticoagulant piscine glycosaminoglycan (i.e.having the pentasaccharide antithrombin binding site), especiallyanticoagulant glucosaminoglycans, and salts and high affinityderivatives thereof.

[0013] Viewed from a further aspect the invention provides a method ofproducing high affinity anticoagulant glycosaminoglycan comprisingextracting endogenous anticoagulant glycosaminoglycan from animalmaterial and optionally depolymerizing and/or molecular weightfractionating said endogenous anticoagulant glycosaminoglycan,characterised in that the animal material is fish material.

[0014] The production of anticoagulant glycosaminoglycans, especiallyglucosaminoglycans, from salmon guts, skin, gills and tail (especiallyguts and gills, in particular gills) is also novel and forms a furtheraspect of the invention. Such anticoagulant glycosaminoglycans,especially high affinity glucosaminoglycans, have particularly goodanticoagulant properties. Thus viewed from a further aspect theinvention provides anticoagulant glycosaminoglycans, and salts andderivatives thereof, extractable from salmon guts, skin, gills or tail,especially guts or gills.

[0015] Viewed from a further aspect the invention provides a method ofproducing anticoagulant glycosaminoglycan comprising extractingendogenous anticoagulant glycosaminoglycan from animal material andoptionally depolymerizing and/or molecular weight fractionating saidendogenous anticoagulant glycosaminoglycan, characterised in that theanimal material is salmon guts, skin, gills or tail, especially guts orgills.

[0016] Viewed from a further aspect the invention provides apharmaceutical composition comprising anticoagulant piscineglycosaminoglycan according to the invention or a salt or derivativethereof together with a physiologically tolerable carrier or excipient,and optionally also a therapeutic or prophylactic drug substance.

[0017] Viewed from a still further aspect the invention provides medicalapparatus, e.g. a catheter, stent or implant, having a surface coated atleast in part with anticoagulant piscine glycosaminoglycan according tothe invention or a salt or derivative thereof.

[0018] Viewed from a still further aspect the invention provides the useof anticoagulant piscine glycosaminoglycan according to the invention ora salt or derivative thereof in mammalian, especially human, medicaltreatment.

[0019] Viewed from another aspect the invention provides anticoagulantpiscine glycosaminoglycan according to the invention or a salt orderivative thereof for use in mammalian medical treatment.

[0020] Viewed from yet another aspect the invention provides a method oftreatment of a human or mammalian body which method comprisesadministering or introducing into said body an anticoagulant effectiveamount of anticoagulant piscine glycosaminoglycan according to theinvention or a salt or derivative thereof.

[0021] The extraction, purification and if desired depolymerization andmolecular weight fractionation of anticoagulant piscineglycosaminoglycan from fish material may be effected analogously to theextraction, purification, etc. of heparin and heparinoids from mammaliansources, e.g. as described in the publications mentioned above. Ifdesired, the fish material used for extraction of glycosaminoglycans maybe subjected to physical or chemical pretreatment, e.g. maceration, acidor base treatment, etc.

[0022] The anticoagulant glycosaminoglycan (anticoagulant GAG) of orused in the invention may be a heparin, a heparinoid, or a low molecularweight heparin or heparinoid, or a mixture of two or more thereof.Preferably it is a sulphated GAG, in particular a heparin or LMWH,especially preferably it contains at least one pentasaccharide unithaving the sequence:

[0023] N-acetylglucosamine-6-O-sulphate,

[0024] glucaronic acid,

[0025] N-sulphated glucosamine-3,6-O-disulphate,

[0026] iduronic acid 2-O-sulphate and

[0027] N-sulphated glucosamine-6-O-sulphate,

[0028] i.e. it is especially preferably a high affinity GAG.

[0029] By “anticoagulant” it is meant that the GAG has the ability tobind to antithrombin, an inter-alpha-trypsin inhibitor, factor Xa, andother proteins to which mammalian heparin binds, e.g. immobilized on asubstrate such as a gel matrix, and/or the ability to delay or preventclotting in human plasma (e.g. increasing clotting time by at least 10%in the test of Example 5 when the GAG is added at 1.0 mg/mL plasma) orto prolong bleeding in a mammal (e.g. a mouse).

[0030] The fish from which the fish material from which the piscine GAGis extracted may be any fish; however fish used as food sources formammals or as raw materials for fish meal, fish food, and fish oil arepreferred. Particularly preferably farmed fish are used. Examples ofsuitable fish include: carp, barbell and other cyprinids; cod, hake,haddock; flounder, halibut, sole; herring, sardine, anchovy; jack,mullet, saury; mackerel, snoek, cutlass fish; red fish; bass; eels (e.g.river eels, conger, etc.); salmon, cod, trout; shad; shark; ray;sturgeon; paddle fish; tilapia and other cichlids; tuna, bonito, billfishes; diadromous fish; etc. Particular examples of suitable fishinclude: flounder, halibut, sole, cod, hake, haddock, bass, jack,mullet, saury, herring, sardine, anchovy, tuna, bonito, bill fish,mackerel, snoek, shark, ray, capelin, sprat, brisling, bream, ling, wolffish, salmon, trout, coho and chinock. Especially preferably the fishused is trout, salmon, cod or herring, more especially salmon.

[0031] The piscine GAG may be used according to the invention in itsnaturally occurring form following extraction. However alternatively itmay be converted into salt form, preferably with a physiologicallytolerable counterion (e.g. sodium, calcium, magnesium, potassium,ammonium or meglumine), or derivatised, e.g. to facilitate its bindingto a surface of an item of medical apparatus, or molecular weightfractionated or depolymerized (e.g. to produce a GAG fraction meetingthe molecular weight definition for LMWH). Such derivatives are likewisepreferably physiologically tolerable. Conventional chemical reactionsmay be used for such derivatization or depolymerization, e.g. reactionswith coupling agents or with molecular weight reducing agents asdescribed in Linhardt et al. supra. Examples of particulardepolymerization techniques include: cleavage with nitrous acid at pH1.5; periodate oxidation followed by cleavage with dilute alkali or mildacid; de-N-acetylation and treatment with nitrous acid at pH 4; andactivation with carbodiimide, hydrazine, aminomethylsulphonate or othercarboxyl activating agents followed by treatment with dilute alkali.

[0032] Coating of items of medical apparatus with piscine GAG may beeffected analogously to coating with mammalian heparin.

[0033] Piscine GAG is chemically distinct from mammalian heparin asdemonstrated by the nmr spectra in FIGS. 1 to 4 of the accompanyingdrawings. FIGS. 1 and 2 are respectively nmr spectra of pig and bovineheparin. FIGS. 3 and 4 are nmr spectra of salmon GAG from intestines andgills respectively. As can be seen, the piscine GAG shows peaks at2.70-2.95 and 3.935 to 3.960 ppm respectively. Viewed from a furtheraspect therefore the invention provides GAG having a peak at 2.70 to2.95 ppm in its ¹H-nmr spectrum in D₂O at 300 MHz and 28° C., inparticular a peak which is at least 10% of the intensity of any peak inthe 2.95 to 3.15 ppm region. Viewed from a further aspect the inventionalso provides GAG having a peak at 3.935 to 3.960 ppm in its ¹H-nmrspectrum in D₂O at 300 MHz and 28° C., in particular a peak which ismore intense than any peak in the 3.960 to 4.100 ppm range. By itsspectrum is here meant the spectrum of the GAG after affinitypurification on an antithrombin-sepharose column (see Example 1hereinafter).

[0034] The piscine GAGs may be separated from mixtures by sequentialprecipitation in various organic solvents (e.g. acetone, methanol,ethanol) or by electrophoretic separation. Alternatively, the GAG groupsmay be separated on anion exchangers (see N.Volpi, J.Chromatography B684:27-34 (1996); Analytical Biochem 240:114-118 (1996);J.Chromatography 622:13-20 (1993); Analytical Biochem 218:382-391(1994)).

[0035] The GAG compositions according to the invention may containnon-GAG components conventional in mammalian GAG compositions, e.g.water (preferably water for injections), ethanol, buffers, osmolalityadjusting agents, preservatives, etc.

[0036] Piscine GAGs may be used in substantially the same quantities asmammalian GAGs are conventionally used or at lower doses as a result ofits anticoagulant efficacy.

[0037] Besides use as anticoagulants, the piscine GAGs of the inventionmay be used as antithrombotics, anti-atherosclerotics, complementinhibitors, anti-inflammatories, anti-cancer agents, anti-viral agents,anti-dementia agents (e.g. anti-Alzheimer agents), anti-prion agents,anti-parasitics, opsonization inhibitors, biomaterials, angiogenesisregulators, and in the treatment of vascular deficit, wounds and immuneresponse disorders (e.g. AIDS), etc. They may be administered enterallyor parenterally, e.g. orally or subcutaneously or bound to an object ordrug material placed into tissue or the circulatory system.

[0038] Besides such therapeutic and surgical uses, the piscine GAGs ofthe invention may be used for diagnostic purposes, e.g. diagnosticsassays, and non-medical uses for which heparin is suited or currentlyused. Thus viewed from a further aspect the invention provides adiagnostic assay kit comprising an anticoagulant, characterised in thatsaid anticoagulant is a piscine glycosaminoglycan according to theinvention.

[0039] The invention extends to piscine GAGs which are notanticoagulants, and their uses, e.g. for purposes for whichnon-anticoagulant mammalian GAGs are suitable. Fareel et al. in RecentAdvances in Blood Coagulation, pages 169-187 (Ed. Poller) have statedthat GAGs, to be useful as antithrombotics, need not necessarily beanticoagulants or react with antithrombin.

[0040] Documents referred to herein are hereby incorporated byreference.

[0041] The present invention will now be illustrated further by thefollowing non-limiting Examples.

EXAMPLE 1 Extraction of Salmon GAG

[0042] Salmon were slaughtered and the intestines were removed and keptcold (ca. −5 to +5° C.) until the following day when they were frozen to−800° C.

[0043] 100 g of the frozen intestines were thawed in 100 mL Tris buffer(0.05M Tris/HCl/0.15M NaCl, pH 7.5) and homogenized in an Ultra Turrax®tissue grinder. The homogenizate was incubated at 80° C. for 1 hour in awater bath, cooled to ambient temperature and centrifuged for 30 minutesat 12000 rpm. The supernatant was removed and the residue washomogenized again in 25 mL of 4M guanidine.HCl in the same buffer. Thiswas again centrifuged as above and the supernatant was removed andcombined with the earlier supernatant. The combined supernatant wasdialyzed against 2×2L of Tris buffer (0.025M Tris/HCl/0.15M NaCl, pH7.5) and centrifuged again as above. Using the Tris buffer used for thedialysis, the resultant supernatant was applied to a column (20 mL bedvolume)of cyanogen bromide activated Sepharose 4B (from Pharmacia) towhich human anti-thrombin was coupled according to the manufacturers'instructions. The same buffer was used to wash the column andsubsequently salmon GAG was eluted using the same buffer at about 0.5mL/min with a linear gradient of salt concentration rising from 0.15M to2.15M NaCl.

[0044] Samples of fractions from the elution were subjected to acarbazole test for heparin and those fractions testing positive werecombined, dialyzed against 5 nM NH₄HCO₃ using a 3500 D mol.wt cut-offmembrane, and freeze dried.

EXAMPLE 2 Extraction of Salmon GAG

[0045] Using the same procedure as Example 1, salmon GAG was extractedfrom 40 g of frozen salmon gills. In the initial incubation, 50 mLrather than 100 mL of extraction buffer was used.

EXAMPLE 3 Antithrombin Binding

[0046] 2.5 mg of the freeze-dried GAG of Example 1 was dissolved per 1mL of a test buffer (comprising Tris buffer (0.05M Tris/2 mMNa₂EDTA/0.18M NaCl), 10 g/L PEG 6000, 0.1 mL/L Tween 80 and HCl to pH8.4) to produce a buffered test sample. 0.2 mL of the test buffer alone(Run 1), or 0.1 mL of the test buffer with 0.1 mL of 3U/mL mammalianheparin (Run 2), or 0.1 mL of the test buffer and 0.1 mL of the testsample (Run 3) were mixed with 0.1 mL bovine thrombin (5U/mL in the testbuffer) and 0.1 mL antithrombin (1500 IU/Ky—Kybernin from Kabi —10 mg/mLin the test buffer), incubated at 37° C. for 5 minutes, mixed with 0.1mL synthetic substrate (S-2238 from Kabi—0.75 mmol/L in water), andincubated at 37° C. for a further 2 minutes. The reaction was stopped byadding 0.1 mL of 50% acetic acid and the amidolytic activity on thesubstrate was measured by determining the absorbance (A) at 405 nm. RunA 1 0.636 (no GAG) 2 0.062 (mammalian GAG) 3 0.228 (piscine GAG)

[0047] The test was repeated with 0, 5 and 20 minutes incubation at 37°C. before synthetic substrate addition. In Runs 4 to 6, 0.6 mL testbuffer were added to 0.3 mL thrombin and 0.3 mL antithrombin, andincubated at 37° C. with 0.4 mL samples being extracted after 0, 5 and20 minutes. The extracts were added to 0.1 mL substrate, incubated at37° C. for 2 minutes, the reaction was stopped and absorbance at 405 nmwas measured. In Runs 7 to 9, 0.3 mL test buffer, 0.3 mL antithrombin,0.3 mL thrombin and 0.3 mL heparin were mixed before the firstincubation/extraction and in Runs 10 to 12 0.3 mL test buffer, 0.3 mLantithrombin, 0.3 mL thrombin and 0.3 mL test sample were mixed beforethe first incubation/extraction. 1st Incubation Run Time (min) A* 4 01.031 (no GAG) 5 5 0.970 (no GAG) 6 20 0.287 (no GAG) 7 0 0.202(mammalian GAG) 8 5 0.086 (mammalian GAG) 9 20 0.213 (mammalian GAG) 100 1.058 (piscine GAG) 11 5 0.221 (piscine GAG) 12 20 0.139 (piscine GAG)

[0048] Runs 1 to 3 were repeated (as Runs 13 to 15) using the piscineGAG of Example 2. Run A 13 0.852 (no GAG) 14 0.070 (mammalian GAG) 150.268 (piscine GAG)

EXAMPLE 4 Clotting Tests

[0049] The piscine GAG of Example 1 was tested for its ability to delayor prevent clotting in human plasma. 2 mg of the freeze dried materialwas dissolved in 0.5 mL 0.05 M tris/HCl/0.1M NaCl buffer, pH 7.4 to givea 4 mg/mL concentration. This was further diluted to give 0.400, 0.200,0.133, 0.080, 0.067 and 0.040 mg/ML samples. 100 μL of each of these wasmixed with 100 μL plasma and 100 μL 3U/mL bovine thrombin and the timeto clotting was measured. As a control (0 mg/mL), 100 μL of the bufferwas used. Concentration (mg/mL) Time* (sec.) 0 20.2 0.040 22.8 0.06725.5 0.080 29.3 0.133 46.8 0.200 >120 0.400 >450

EXAMPLE 5 Clotting Test

[0050] Using the Cephotest (Nycomed, Oslo) on a StaCompact (Stago,France) apparatus the effect of the piscine GAG of Example 1 on plasmaclotting time was measured.

[0051] Piscine GAG of Example 1 was dissolved in Owren-Koller buffer toconcentrations of 0.08 to 10.0 mg/mL. These were diluted in a 9:1 volumeratio with pooled human plasma and the clotting times determined.Piscine GAG Clotting Time concentration (mg/mL) (seconds) 0 28.1 0.00828.5 0.016 29.8 0.033 31.4 0.067 34.1 0.125 41.6 0.250 58.4 0.500 96.90.750 134.9 1.000 178.7

EXAMPLE 6 Activity in Heparin Test

[0052] 6 mg of the piscine GAG of Example 1 was dissolved in 0.6 mLOwren Koller buffer to give a 10 mg/mL primary solution. Samples of thiswere diluted with Owren Koller buffer (from Stago, France) to give 5mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.67 mg/mL, 0.33 mg/mL, 0.16 mg/mL and0.08 mg/mL secondary samples. These were tested for their “heparinactivity” using a standard Stachrom LMWH kit from Stachrom. Since thetest system involves addition of test sample and antithrombin to plasma,the results are independent of the antithrombin content of the plasma.The ‘heparin’ content of the piscine GAG was determined in U/mL relativeto standard heparin samples (Hepanorm from Stago), giving an averagevalue of “heparin activity” U/mg for the piscine GAG as anti Xa of 1.39.

EXAMPLE 7 NMR-Spectra of GAGs

[0053] Bovine heparin and porcine heparin (sodium salts, H-0777 andH-9399 from Sigma—isolated from intestinal mucosa) were purified usingan antithrombin-Sepharose column as in Example 1.

[0054] Virtually salt free samples of the purified bovine and porcineheparins and the salmon GAG of Examples 1 and 2 were freeze-dried anddissolved in D₂O. Tris was added as a marker. The ¹H-nmr spectra werethen recorded at 28° C. using a Varian 300 MHz nmr spectrometer. Thespectra are shown in FIGS. 1 to 4. In these the major peaks at 4.65 and3.5 ppm are water and Tris respectively.

[0055] Recorded peaks are set out in Table 1 below. TABLE 1 Sample Peaks(ppm) Bovine 0.994 1.854 3.429 3.438 3.452 3.490 3.521 3.534 3.558 3.5833.594 3.610 3.615 3.629 3.662 3.665 3.713 3.727 3.812 3.826 3.837 3.8463.853 3.884 3.906 3.929 3.936 3.938 3.945 4.017 4.052 4.138 4.141 4.4254.492 4.515 4.559 4.603 4.627 4.692 5.032 5.224 Porcine 1.821 3.0493.072 3.247 3.319 3.337 3.359 3.490 3.674 3.682 3.729 3.792 3.811 3.8613.992 4.121 4.174 4.452 4.773 5.011 5.149 Salmon 0.759 1.140 1.163 1.2271.249 intestine 1.739 1.865 3.378 3.389 3.423 3.451 3.490 3.535 3.6003.610 3.646 3.728 3.774 3.794 3.951 4.429 4.496 4.518 4.562 4.607 4.6284.696 4.714 Salmon 0.758 0.996 1.020 1.047 1.128 gills 1.151 1.167 1.2181.243 1.279 1.304 1.489 1.728 1.853 1.925 2.820 3.015 3.168 3.243 3.2473.339 3.346 3.359 3.367 3.376 3.400 3.406 3.424 3.440 3.446 3.486 3.4903.523 3.534 3.580 3.596 3.604 3.618 3.634 3.722 3.771 3.783 3.811 3.9273.942 4.152 4.182 4.205 4.215 4.222 4.246 4.265 4.288 4.314 4.360 4.4264.492 4.514 4.559 4.603 4.623 4.668 4.691

[0056] The peaks at 3.86, 5.14 and 5.01 ppm in bovine and porcineheparin are not present for salmon GAG. The peaks at 4.12 and 3.99 ppmin bovine and porcine heparin are weak or absent in salmon GAG.

[0057] The peaks at 2.8-2.85 and 3.940-3.955 ppm in salmon GAG areabsent in porcine and bovine heparin as are the peaks at 3.01, 3.16,3.24, 3.33, 3.34, 4.28 and 4.31 ppm.

EXAMPLE 8 GAG Extraction from Salmon Intestine

[0058] 445 g of salmon intestines were mixed with 400 mL water andhomogenized. The homogenate was incubated at 80° C. for 1 hour and thenrehomogenized. The homogenate was centrifuged at 12,000 rpm for 30minutes and the solid phase discarded. The pH of the supernatant wasadjusted to approx. 8.9 using NaOH and kept at 90° C. for 15 minutesbefore being centrifuged again as above. This supernatant was applied toa column with the anion exchanger Dowex 2 using a bed volume around415mL. The column was washed with 5 mM NH₄HCO₃ and the pH adjusted to8.9 with NH₃ and 0.1M NaCl. The column was then washed with the samebuffer containing 1.6M NaCl.

[0059] Elution was performed using the same buffer containing 2.4M NaCl.The eluate was shown to contain uronic acid using the carbazole test.The eluate from using 1.6M NaCl was also shown to contain uronic acidusing the same test.

[0060] An aliquot of 10 mL of the GAG eluate using 2.4M NaCl wasdialyzed against water, freeze dried, dissolved in 5 mL water and testedfor antithrombin accelerating activity:

[0061] The test buffer used was 0.1M Tris and HCl to pH 8.4.

[0062] 0.1 mL thrombin (bovine plasma, Sigma, 5 U/mL in test buffer) wasadded to 0.3 mL test buffer alone (Run 1) or 0.2 mL test buffer and 0.1mL water (Run 2) or 0.2 mL test buffer and 0.1 mL antithrombin(Atenativ, Pharmacia & Upjohn, 0.1 mg/mL in test buffer) (Run 3) or 0.1mL test buffer and 0.1 mL antithrombin (0.1 mL in test buffer) and 0.1mL dissolved 2.4 M NaCl eluate (Run 4). Each mixture was further mixedwith 0.1 mL synthetic sustrate (S-2238 from Kabi—0.75 mmol/L in water)and incubated at 37° C. for 2 minutes. The reaction was stopped byadding 0.1 mL of 20 % acetic acid and the amidolytic activity on thesubstrate was measured by determining the absorbance (A) at 405 nm.Results from the 2.4 M NaCl fraction Run A, 405 nm Comments 1 1.070 noGAG 2 1.028 no GAG 3 0.761 thrombin & antithrombin 4 0.279 thrombin &antithrombin & piscine GAG

[0063] An aliquot of 10 mL of the 1.6 M NaCl eluate was dialyzed againstwater, freeze dried and dissolved in 5 mL water and tested forantithrombin accelerating activity using the solutions and reagentsdescribed in run above:

[0064] 0.2 mL test buffer and 0.1 mL thrombin and 0.1 mL antithrombin(Run 1) or 0.1 mL test buffer and 0.1 mL thrombin and 0.1 mLantithrombin and 0.1 mL dissolved 1.16M NaCl eluate (Run 2). These wereeach mixed with 0.1 mL synthetic substrate (S-2238 from Kabi—0.75 mmol/Lin water) and incubated at 37° C. for 2 minutes. The reaction wasstopped by adding 0.1 ML of 20% acetic acid and the amidolytic activityon the substrate was measured by determining the absorbance (A) at 405nm. Results from the 1.6 M NaCl fraction Run A, 405 nm Comments 1 0.767thrombin & antithrombin 2 0.351 thrombin & antithrombin & piscine GAG

[0065] In both cases, that is from both the 1.6 M NaCl fraction and fromthe 2.4 M NaCl fraction (runs 2 and 4 respectively) there areanticoagulation effects from piscine GAG.

1. Anticoagulant piscine gill glucosaminoglycans and salts andderivatives thereof.
 2. High affinity anticoagulant piscineglycosaminoglycans and salts and high affinity derivatives thereof. 3.Anticoagulant glycosaminoglycans, and salts and derivatives thereof,extractable from salmon guts, skin, tail or gills.
 4. Glycosaminoglycanhaving a peak at 2.70 to 2.95 ppm in its ¹H-nmr spectrum in D₂O at 300MHz and 28° C.
 5. Glycosaminoglycan having a peak at 3.935 to 3.960 ppmin its ¹H-nmr spectrum in D₂O at 300 MHz and 280C.
 6. Anticoagulantglucosaminoglycans and salts and derivatives thereof as claimed in anyone of claims 1 to 5 in sterile form.
 7. Anticoagulantglucosaminoglycans and salts and derivatives thereof as claimed in anyone of claims 1 to 6 containing no more than 10% by weight ofnon-glucosaminoglycan biological materials.
 8. Anticoagulantglucosaminoglycans and salts and derivatives thereof as claimed in anyone of claims 1 to 6 containing no more than 2% by weight ofnon-glucosaminoglycan biological materials.
 9. Anticoagulantglucosaminoglycans and salts and derivatives thereof as claimed in anyone of claims 1 to 8 having a molecular weight in the range 500 to30,000 Daltons.
 10. Anticoagulant glucosaminoglycans and salts andderivatives thereof as claimed in any one of claims 1 to 8 having amolecular weight of at least 1000 Daltons.
 11. Anticoagulantglucosaminoglycans and salts and derivatives thereof as claimed in anyone of claims 1 to 8 having a molecular weight of at least 1500 Daltons.12. Anticoagulant glycosaminoglycans and salts and derivatives thereofas claimed in any one of claims 1 to 11 containing at least onepentasaccharide unit having the sequenceN-acetylglucosamine-6-O-sulphate, glucoronic acid, N-sulphatedglucosamine-3,6-O-disulphate, iduronic acid 2-O-sulphate and N-sulphatedglucosamine-6-O-sulphate.
 13. A pharmaceutical composition comprising ananticoagulant piscine glycosaminoglycan or a salt or derivative thereofas claimed in any one of claims 1 to 12 together with a physiologicallytolerable carrier or excipient, and optionally also a therapeutic orprophylactic drug substance.
 14. Medical apparatus having a surfacecoated at least in part with anticoagulant piscine glycosaminoglycan ora salt or derivative thereof as claimed in any one of claims 1 to 12.15. The use of anticoagulant piscine glycosaminoglycan or a salt orderivative thereof as claimed in any one of claims 1 to 12 in mammalianmedical treatment.
 16. Anticoagulant piscine glycosaminoglycans andsalts and derivatives thereof as claimed in any one of claims 1 to 12for use in mammalian medical treatment.
 17. A method of treatment of ahuman or mammalian body which method comprises administering orintroducing into said body an anticoagulant effective amount ofanticoagulant piscine glycosaminoglycan or a salt or derivative thereofas claimed in any one of claims 1 to
 12. 18. A method of producinganticoagulant glycosaminoglycans comprising extracting endogenousanticoagulant glycosaminoglycan from animal material and optionallydepolymerizing and/or molecular weight fractionating said endogenousanticoagulant glycosaminoglycan, characterised in that the animalmaterial is fish gills.
 19. A method of producing anticoagulantglycosaminoglycans comprising extracting endogenous anticoagulantglycosaminoglycan from animal material and optionally depolymerizingand/or molecular weight fractionating said endogenous anticoagulantglycosaminoglycan, characterised in that the animal material is salmongills, skin, tail, guts or a mixture thereof.
 20. A method as claimed inclaim 18 or claim 19 wherein said animal material is salmon gills.
 21. Adiagnostic assay kit comprising an anticoagulant, characterised in thatsaid anticoagulant is a piscine glycosaminoglycan as claimed in any oneof claims 1 to 12.